Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind

Vaccinia E3 protein has the biochemical capacity of binding to double-strandedRNA (dsRNA). The best characterized biological func-tions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNAbinding capacity in the biological functions of the E3 protein is not clear. To further understand themechanismof the E3 protein biological functions, we performed alanine scanning of the entire dsRNAbinding domain of the E3 protein to examine the link between its biochemical capacity of dsRNAbinding andbiological functions.Of the 115mutants examined, 20were defective in dsRNAbinding. Although themajority of themutants defective in dsRNAbinding also showeddefec-tive replication inHeLa cells, ninemutants (I105A, Y125A, E138A, F148A, F159A,K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3Lmutants showed that residues essential for dsRNAbinding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, sup-pression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novelmechanismwhich does not correlatewith its dsRNAbinding activity. IMPORTANCE dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antivi-ral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved b